Introduction of foreign genes into eukaryotic cells (transfection) is a commonly-used technique in molecular biology and cell biology laboratories. There are two different types of transfection: (1) transient transfection where introduced genes are studied for a short period of time; or (2) stable transfection where genes are introduced permanently into cell lines. The stable transfection process requires a selection of the transfected cells for further replication and studies.
The transfection process commences with an initial growth of a culture of eukaryotic cells in a liquid medium in a culture dish. A foreign DNA, with which the eukaryotic cells are to be transfected, is prepared using a calcium phosphate coprecipitation method or another equivalently effective method, to obtain a DNA precipitate. The liquid medium is removed from the culture dish and the foreign DNA precipitate is added to the eukaryotic cells. At the same time, a marker DNA precipitate is added that will enable subsequent selection of the Eukaryotic cells that uptake the DNA inclusions. In general, the marker DNA includes a gene that codes for an enzyme that will provide resistance to a drug for the cells which uptake the marker DNA (and the foreign DNA). For example, if the drug Neomycin will be used to select the transfected cells, then a Neomycin resistant gene will be used as the marker DNA during transfection.
Once the cells have had both the marker and foreign DNA precipitates added to the cell culture, the cell culture is allowed to grow for a period of time. Then, the cells are fed with a medium containing an appropriate drug. Only those cells that took up the marker DNA (and foreign DNA) are resistant to the toxic effect of the drug and grow in its presence. Non-transfected cells die. This procedure takes approximately 15-20 days. Once the non-transfected cells are dead, the growth of the transfected cells can be visualized as distinct small groups of cells (clones) in the culture dish. The clones now must be transferred to another dish for replication and further study.
The conventional method for segregation of transfected cell clones is illustrated in FIG. 1. A culture dish 10 includes a culture medium 12 in which clones 14 have been grown, after transfection. Initially, the laboratory technician views culture dish 10 from below (as indicated by arrow 16) and employing a felt marker, marks each of the clones on the bottom of culture dish 10 so as to enable identification of the clones from above. Next, culture medium 12 is removed and a cloning ring 18 (which is a glass cylinder having a silicone grease coating at least about its lower aperture) is placed over a colony and is affixed firmly to the bottom of culture dish 10. A trypsin solution is then added into the well of cloning ring 18 and causes the cells of the clone to separate from the bottom of culture dish 10. The grease coating on the bottom of cloning ring 18 prevents leakage of the trypsin solution from within the cloning ring's well. At this point, the lab technician aspirates the trypsin/clone from the well and places the aspirant into another dish containing medium, for further growth.
The above described procedure exhibits a number of disadvantages which make the process both time consuming and inefficient in the recovery of cloned cells. In order to fix cloning ring 18 to the bottom of culture dish 10, the culture medium is first removed. The cell clones, as a result, immediately start to dry. After a few cloning rings have been affixed and cells of a few clones removed, the remaining clones on the dish often have dried out and have become useless. The trypsin solution added to a cloning ring 18 may leak out if the cloning ring does not make a good seal with culture dish 10. Further, the trypsin solution is not completely effective in removing all of the cells of a clone from the bottom of culture dish 10, so there are some cloned cells that are not recoverable. Finally, cloning rings are expensive and require organic solvents for cleaning. Disposal of such solvents is a continuing problem.
Accordingly, it is an object of this invention to provide an improved method for transfected cell selection.
It is another object of this invention to provide a rapid method for transfected cell selection that eliminates a need for cloning rings.
It is yet another object of this invention to provide an apparatus for transfected cell selection that enables improvements in the recovery of stably transfected cells.